Environmental and endogenous exposure to chemicals that produce DNA-protein and DNA-peptide crosslinks are correlated with an increased risk of cancer, asthma, and other diseases. These exposures take place in both occupational and in-home settings and affected individuals often experience multi-year chronic exposures that are far in excess of typical indoor air quality standards. One of the most common DNA-protein crosslink-inducing agents is formaldehyde and currently thousands of individuals in the US population are exposed to this and other aldehydic compounds. To understand the biological processing of these DNA lesions, our laboratories have i) established synthetic chemical procedures to create DNAs containing site-specifically modified DNA-protein crosslinks, ii) identified DNA polymerases that are capable of catalyzing translesion synthesis of these lesions, and iii) carried out dose-dependent, genome-wide assays that identified genes whose products function to limit DNA-protein crosslink- induced cytotoxicity. These investigations have generated a series of hypotheses which postulate that when eukaryotic cells are exposed to chronic, low levels of DNA-protein crosslinking agents, cytotoxicity and mutagenesis are minimized by tolerance pathways involving homologous recombination. However, following acute high dose exposures, it is hypothesized that cells will shift to pathways involving components of either nucleotide excision repair or translesion synthesis. To address these hypotheses, gene-specific deletions or siRNA depletion and small molecular inhibitors will be used to identify the constellation of genes and interrelated pathways that are critical in limiting cellular toxicity and mutagenesis. Possible roles of individual gene products in modulating cellular responses to DNA-protein crosslinks may include involvement in DNA repair, recombination, translesion synthesis, cell cycle check points, and proteolytic pathways. Biochemical analyses of repair intermediates and the activities of helicases and translesion synthesis polymerases will be established using DNAs containing site-specific DNA-peptide and protein crosslinks. Collectively, these investigations will yield comprehensive analyses of repair and tolerance of this class of DNA lesions.